This proposal is focused on understanding the molecular composition, mechanism of action, and modes of communication of mammalian endoplasmic reticulum (ER)-organelle membrane contact sites (MCSs). Increasing evidence indicates that ER MCSs are crucial regulatory hubs, but they are poorly defined on both the molecular and functional levels. I will address this deficit by examining the mammalian homologs of a newly described class of conserved ER MCS proteins, termed Loc (lipid transfer at organelle contact site), identified in our lab in yeast (yLoc). We have demonstrated that in vivo yLocs directly facilitate membrane contacts between the ER and many other organelles and in vitro yLocs mediate the non-vesicular sterol transport between liposomes. My preliminary data reveal that the mammalian Loc (mLoc) family member GRAMD2 localizes to Ca2+-dependent ER-plasma membrane (PM) MCSs, which function to regulate store-operated Ca2+ entry (SOCE). SOCE is a PM Ca2+ influx pathway that is induced by depletion of Ca2+ from ER stores and is critical for cell homeostasis. Building on this observation and our yLoc data, I will focus on understanding the molecular basis and functions of mLocs at ER MCSs. Specifically, in Aim 1, I will examine the functional role of GRAMD2 at Ca2+-regulated ER-PM contacts. In Aim 2, I will define molecular activities and the mechanistic role of GRAMD2. In Aim 3, I will probe the functional role of other mLoc protein family members and examine their relationships at ER MCSs. Understanding the fundamental role of mLoc will inform upon MCSs regulation and composition will provide fundamental insight into the roles of ER MCSs in cell physiology, which will improve our understanding of the etiology human disease and could reveal new targets for therapeutics.